ADAM19: a Novel Target for Metabolic Syndrome in Humans and Mice

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ADAM19: A Novel Target for Metabolic Syndrome in Humans and Mice

Lakshini Weerasekera

Caroline Rudnicka

Qing-Xiang Sang

Joanne E. Curran The University of Texas Rio Grande Valley

Matthew P. Johnson The University of Texas Rio Grande Valley

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Recommended Citation Weerasekera, L., Rudnicka, C., Sang, Q.-X., Curran, J. E., Johnson, M. P., Moses, E. K., Göring, H. H. H., Blangero, J., Hricova, J., Schlaich, M., & Matthews, V. B. (2017). ADAM19: A Novel Target for Metabolic Syndrome in Humans and Mice. Mediators of Inflammation, 2017, 7281986. https://doi.org/10.1155/ 2017/7281986

This Article is brought to you for free and open access by the School of Medicine at ScholarWorks @ UTRGV. It has been accepted for inclusion in School of Medicine Publications and Presentations by an authorized administrator of ScholarWorks @ UTRGV. For more information, please contact [email protected], [email protected]. Authors Lakshini Weerasekera, Caroline Rudnicka, Qing-Xiang Sang, Joanne E. Curran, Matthew P. Johnson, Eric K. Moses, Harald H. H. Goring, John Blangero, Jana Hricova, Markus Schlaich, and Vance B. Matthews

This article is available at ScholarWorks @ UTRGV: https://scholarworks.utrgv.edu/som_pub/123 Hindawi Mediators of Inﬂammation Volume 2017, Article ID 7281986, 9 pages https://doi.org/10.1155/2017/7281986

Research Article ADAM19: A Novel Target for Metabolic Syndrome in Humans and Mice

Lakshini Weerasekera,1 Caroline Rudnicka,2 Qing-Xiang Sang,3 Joanne E. Curran,4 Matthew P. Johnson,4 Eric K. Moses,5 Harald H. H. Göring,4 John Blangero,4 Jana Hricova,1 Markus Schlaich,1,2 and Vance B. Matthews1

1 School of Medicine and Pharmacology, University of Western Australia, Crawley, Western Australia, Australia 2Royal Perth Hospital, Perth, Australia 3Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, USA 4South Texas Diabetes & Obesity Institute, University of Texas Rio Grande Valley School of Medicine, Brownsville, TX, USA 5Centre for Genetic Origins of Health & Diseases, Faculty of Medicine, Dentistry & Health Sciences, University of Western Australia and Faculty of Health Sciences, Curtin University, Western Australia, Australia

Correspondence should be addressed to Vance B. Matthews; [email protected]

Received 13 October 2016; Accepted 12 January 2017; Published 7 February 2017

Academic Editor: Julio Galvez

Copyright © 2017 Lakshini Weerasekera et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Obesity is one of the most prevalent metabolic diseases in the Western world and correlates directly with insulin resistance, which may ultimately culminate in type 2 diabetes (T2D). We sought to ascertain whether the human metalloproteinase A Disintegrin and Metalloproteinase 19 (ADAM19) correlates with parameters of the metabolic syndrome in humans and mice. To determine the potential novel role of ADAM19 in the metabolic syndrome, we first conducted microarray studies on peripheral blood mononuclear cells from a well-characterised human cohort. Secondly, we examined the expression of ADAM19 in liver and gonadal white adipose tissue using an in vivo diet induced obesity mouse model. Finally, we investigated the effect of neutralising ADAM19 on diet induced weight gain, insulin resistance in vivo, and liver TNF-𝛼 levels. Significantly, we show that, in humans, ADAM19 strongly correlates with parameters of the metabolic syndrome, particularly BMI, relative fat, HOMA-IR, and triglycerides. Furthermore, we identified that ADAM19 expression was markedly increased in the liver and gonadal white adipose tissue of obese and T2D mice. Excitingly, we demonstrate in our diet induced obesity mouse model that neutralising ADAM19 therapy results in weight loss, improves insulin sensitivity, and reduces liver TNF-𝛼 levels. Our novel data suggest that ADAM19 is pro-obesogenic and enhances insulin resistance. Therefore, neutralisation of ADAM19 may be a potential therapeutic approach to treat obesity and T2D.

1. Introduction proteins, in metabolic disorders. ADAMs are proteolytic enzymes which regulate cell phenotype through affecting Obesity is one of the most prevalent metabolic diseases in cell adhesion, migration, proteolysis, and signalling [2]. Our the Western world and correlates with glucose intolerance, group was the first to identify that ADAM28 is elevated in insulin resistance, dyslipidemia, and cardiovascular disease. humans with the metabolic syndrome and is a novel sheddase These metabolic parameters may ultimately culminate in of human tumour necrosis factor-𝛼 (TNF-𝛼)[3].Relevantly, pancreatic beta cell failure and type 2 diabetes (T2D) [1]. TNF-𝛼 is a major proinflammatory cytokine implicated in the As the financial and social burden of obesity escalates, it is metabolic syndrome [4, 5] and it is well established that TNF- crucial to develop new human therapeutics to alleviate the 𝛼 induces insulin resistance [6, 7]. Similarly, the activity of adverseconsequencesofthemetabolicsyndrome. ADAM17, also known as TNF-𝛼 converting enzyme (TACE), There has been growing interest in the role of metallo- correlateswithinsulinresistance[8,9]andadministration proteinases or A Disintegrin and Metalloproteinase (ADAM) of TACE inhibitors to humans in clinical trial settings has 2 Mediators of Inflammation proven to effectively decrease inflammatory mediators [10]. weeks. Body weights were recorded on a weekly basis. At Interestingly, our group and others have shown that TNF- the end of the experiment, mice were sacrificed and the liver 𝛼 is also a substrate for ADAM19 [11–13] and therefore it is andgonadalwhiteadiposetissuewascollectedforparaffin possiblethatADAM19mayhaveanunderlyingroleinthe embedding for immunohistochemistry and snap frozen in pathogenesis of obesity and T2D. liquid nitrogen for mRNA studies. ADAM19, also known as meltrin 𝛽, was identified and In our second cohort of mice, eight-week-old male characterised by our team [14, 15] and others [16]. ADAM19 specific pathogen-free C57BL6/J mice were obtained from the contains several complex domains, including a pro-domain, Animal Resources Centre (ARC, Perth). Mice were placed on metalloproteinase domain, disintegrin domain, cysteine-rich different diet/antibody treatment regimes: (1) standard chow domain, epidermal growth factor-like domain, transmem- (𝑛=9); (2) high fat diet, administered rabbit pre-immune brane domain, and cytoplasmic tail domain [15]. Of most IgG (𝑛=3); (3) high fat diet, administered rabbit ADAM19 relevance, the metalloproteinase domain of ADAM19 is neutralising antibody (𝑛=3). Neutralising antibodies were knowntobeinvolvedinextracellularmatrixbreakdownand administered at week 10 of the dietary regiment as ADAM19 reconstruction [14]. One of the most important functions protein is increased at this time-point in tissues such as liver. carried out by the metalloproteinase domain of ADAM19 Mice received 100 𝜇g of antibody every 2 days via tail vein is the catalytically mediated ectodomain shedding of sub- for the final two weeks of feeding. Insulin tolerance tests strates[14]suchasTNF-𝛼. ADAM19 is expressed in numer- (ITT; 0.5 U/kg) and glucose tolerance tests (GTT; 1 g/kg) were ous metabolically relevant cell types and tissues, including performed at the end of week 12. Body weight measurements peripheral blood mononuclear cells, liver, and skeletal muscle were determined each week. Mice were sacrificed at the end [14]. Furthermore, ADAM19 has been linked to numerous of the experiment and their blood was collected for serum diseases [15] and serves important biological functions in and livers were collected for paraffin embedding. embryogenesis [17], cardiovascular system development [18], and skeletal muscle adaptation [19]. Our study is the first to 2.3. ADAM19 Neutralising Antibody. The rabbit ADAM19 examine the role of ADAM19 in obesity, insulin resistance, neutralising antibody [pAb361] used for our experiments is and T2D. well characterised by our team [21, 22]. In this study, we hypothesised that ADAM19 is associated with the metabolic syndrome in humans and mice. The out- 2.4. ADAM19 Immunohistochemistry on Murine Liver. Liver comes of this study provide novel insights into the ADAM19- tissue of mice fed with standard chow or HFD was fixed mediated pathogenesis of obesity, insulin resistance, and in 4% paraformaldehyde and paraffin embedded. Standard T2D. immunohistochemistry procedures were conducted, including antigen retrieval and incubations with primary antibody 2. Materials and Methods (rabbit anti-hADAM19 disintegrin domain IgG (pAb362)) 2.1. Human Microarray for the Identification of Metallopro- at a 1:200 dilution [21, 23]. Sections were visualised with teinases Involved in Obesity and T2D. RNA samples (from diaminobenzidine (DAB; DAKO). peripheral blood mononuclear cells) were obtained from the 𝛼 San Antonio Family Heart Study (SAFHS) (𝑛 = 1240), a 2.5. Adam19 and Tnf- mRNA Expression Studies in Livers study of risk factors for cardiovascular disease in Mexican and/orGonadalWhiteAdiposeTissueofMiceFedaHFD. Americans living in and around San Antonio, Texas [20]. RNA from livers of mice fed a HFD was extracted using Trizol The SAFHS is a large family-based genetic epidemiological reagent (Invitrogen) and cDNA synthesis was performed study including 1431 individuals from 42 extended families using the High Capacity RNA-to-cDNA kit (Applied Biosys- at baseline. Individuals from large randomly selected, multi- tems). Real-time PCR to determine the mRNA abundance of 𝛼 generational pedigrees were sampled independent of their mouse Tnf- and Hprt (house-keeper gene) was performed phenotype or the presence or absence of disease. All partic- using a Rotor-gene real-time PCR machine (Qiagen) using ipants in the SAFHS provided informed consent. The study predeveloped TaqMan probe (FAM labelled) and primer 𝛼 and all protocols were approved by the Institutional Review sets for mouse Tnf- (Mm00443260 g1), mouse Adam19 Board at the University of Texas Health Science Centre at San (Mm00477337 m1), and mouse Hprt (Mm01545399 m1) Antonio (San Antonio, TX). Gene expression profiles were (Applied Biosystems). Quantitation was conducted as previ- generated using microarrays, and data processing steps and ously described [24]. quality control are as described previously [20]. 2.6. TNF-𝛼 ELISA on Murine Liver Protein. Liver tissue 2.2. Animals. In our first mouse cohort, eight-week-old male was homogenised in cytosolic extraction buffer containing specific pathogen-free C57BL6/J mice were obtained from phosphatase and protease inhibitors. Protein levels were the Animal Resources Centre (ARC, Perth). Mice were determined using a Bradford protein assay and TNF-𝛼 administered a normal chow (14.3 MJ/kg, 76% of kJ from was measured in lysates using a mouse TNF-𝛼 ELISA carbohydrate, 5% from fat, 19% from protein; Specialty Feeds, (http://www.elisakit.com/). Glen Forrest, WA, Australia) or high fat diet, HFD (19 MJ/kg, 35% of kJ from carbohydrate, 42% from fat, 23% from 2.7. Statistics. Associations between ADAM19 lymphocyte- protein; Specialty Feeds, Glen Forrest, WA, Australia) for 12 derived gene expression and clinical parameters in the Mediators of Inflammation 3

Table 1: Parameters of the metabolic syndrome are associated with 3.2. ADAM19 and Its Substrate TNF-𝛼 Are Elevated in Liver high ADAM19 expression in human peripheral blood mononuclear Biopsies from Our HFD-Fed Mice. To complement our clini- cells (𝑛 = 1240). cal observations, we explored ADAM19 expression and activity in animal models. Mice were weighed weekly to confirm Parameter 𝑝 value Direction of correlation obesity (Figure 1(a)). T2D was verified by insulin tolerance Fasting insulin 0.003 Positive tests and glucose tolerance tests at the end of the experiment BMI 0.000046 Positive (Supplementary Figure 3 in Supplementary Material available HDL cholesterol 0.000065 Negative online at https://doi.org/10.1155/2017/7281986) as previously conducted by our group [25]. We next investigated ADAM19 Relative fat 0.000017 Positive protein expression in liver, as it is an organ that is involved HOMA-IR 0.00014 Positive inlipidandglucosemetabolism[26].Ithasbeenpreviously TNF-𝛼 0.018 Positive reported that there is low expression of ADAM19 in murine Triglycerides 0.05 Positive liver[16].WefoundthatADAM19proteinlevelsareincreased C-reactive protein 0.05 Positive in the highly steatotic livers of mice that have HFD-induced obesity and T2D compared to mice fed a normal chow 𝛽 IL-1 0.05 Positive (Figures 1(b) and 1(c)). BMI: body mass index; HDL: high-density lipoprotein; TNF: tumor necrosis As previously mentioned, TNF-𝛼 is a proinflammatory factor; IL: interleukin. cytokine involved in the metabolic syndrome and is a known substrate for ADAM19. Given that ADAM19 gene expression in human lymphocytes correlates with TNF-𝛼 protein level in the blood stream (Table 1) and ADAM19 protein is elevated Mexican American pedigrees were determined using a lin- in the liver of mice fed a HFD, we also wanted to determine ear mixed model allowing for residual non-independence whether Tnf-𝛼 gene expression is increased in the livers of amongst family members as a function of their genetic ourcohortofmicefedaHFD.WefoundthatmRNAlevelsof relationships. A likelihood ratio statistic was utilised to Tnf-𝛼 are elevated in liver (Figure 2) biopsies from our mice test whether ADAM19 expression significantly predicted a that have HFD-induced obesity and T2D compared to chow relevant standard clinical parameter (fasting insulin, BMI, fed mice. In addition, we also highlighted that both Adam19 HDL-C, relative fat mass, HOMA-IR index of insulin resis- (Figure 3(a)) and Tnf-𝛼 (Figure 3(b)) gene expression were tance, TNF-𝛼, triglycerides, C-reactive protein, and IL-1𝛽). increased in gonadal white adipose tissue from HFD mice Other primary covariates were simultaneously controlled for compared to chow fed mice. including sex and age. All analyses were performed using the statistical package, SOLAR. 3.3. Neutralising ADAM19 Causes Weight Loss in HFD-Fed All in vivo results are expressed as the mean + and/or − Mice. We are the first group to assess the effect of neutralising standard error of the mean (SEM). Data were analysed for ADAM19 activity in vivo on the parameters of the metabolic differences by Student's 𝑡-test for unpaired samples where syndrome. Through our ADAM19 neutralisation experiment, appropriate. Data was considered to be statistically significant we found in our obese and T2D mice that neutralising when 𝑝 ≤ 0.05. ADAM19 activity minimises the accumulation of gonadal adiposetissuecomparedwithmiceadministeredtheIgG 3. Results control antibody (Figure 4). Secondly,weshowedforthefirsttimethatHFD-fed 3.1. ADAM19 Expression in Human Blood Mononuclear Cells mice administered ADAM19 neutralising antibody lost a Is a Novel Marker of the Metabolic Syndrome. Here we use significant amount of weight when compared with mice that a large human cohort, the San Antonio Family Heart Study were administered rabbit IgG control antibody injections (SAFHS), which is well characterised for T2D and cardio- (𝑝 < 0.02) (Figure 5). Mice were monitored daily during vascular disease, that has also been extensively genetically antibody administrations and both groups displayed normal analysed and carries full microarray-based genome wide gene behaviour, posture, response to touch, and healthy coats. This expression profiles on 1,240 individuals. This dataset was novel finding provides evidence that neutralising antibody filtered to identify novel metalloproteinases involved in the directed against the metalloproteinase domain of ADAM19 development of obesity and type 2 diabetes. We have found couldbeapotentialtherapeutictoalleviatesymptomsofthe that high-level expression of ADAM19 in blood mononu- metabolic syndrome. clear cells from the SAFHS cohort correlated strongly with parameters of the metabolic syndrome and in particular 3.4. Neutralising ADAM19 Improves Insulin Sensitivity in BMI, relative fat, index of insulin resistance (HOMA-IR), and HFD-Fed Mice. Given that obese and T2D mice adminis- triglycerides (Table 1). In addition, ADAM19 expression in tered ADAM19 neutralising antibody lost weight, we were the SAFHS cohort also strongly correlated with circulating C- curioustoexploreifthesemicewerealsomoreinsulin reactive protein, IL-1𝛽,andTNF-𝛼 levels (Table 1). The latter sensitive. We performed an insulin tolerance test (ITT) at is relevant as TNF-𝛼 is a substrate for ADAM19. These highly the end of the experiment. Despite the ADAM19 neutralising significant clinically relevant observations suggest a role for antibody group commencing the experiment with higher ADAM19 in the regulation of human metabolism. fasting glucose compared to the rabbit IgG group, the group 4 Mediators of Inflammation

150 ∗ 145 ∗ ∗ ∗ 140 ∗ ∗ 135 ∗ ∗ 130 ∗ 125 ∗ 120 ∗ 115 ∗ 110 105 100 % increase from starting body% increase from weight 0123456789101112 Time (weeks)

HFD Standard chow (a)

(b) (c)

Figure 1: High fat diet induces obesity and elevates ADAM19 expression in mice. Mice fed a high fat diet gained weight (a); 𝑛=14–18/group. Representative photomicrographs of ADAM19 protein expression in livers from mice fed either a normal chow (b) or high fat diet (c). ∗ ADAM19 staining is cytoplasmic and brown in colour. 200x magnification. 𝑝 < 0.05.

∗ 3 3.5. Neutralising ADAM19 Reduces TNF-𝛼 Protein Levels but Does Not Effect ADAM19 Protein Expression in Livers from HFD-Fed Mice. As ADAM19 is a sheddase of TNF-𝛼 protein, 𝛼 2 we measured the TNF- protein levels by ELISA in the livers of mice treated with either rabbit IgG control antibody or ADAM19 neutralising antibody. We found that neutralising 𝛼

mRNA expression mRNA ADAM19 activity resulted in reduced TNF- protein levels

𝛼 1 in the liver (Figure 7). The TNF-𝛼 protein may be cleaved or cytoplasmic derived TNF-𝛼. TNF-

(fold change relative to chow) to relative change (fold We also examined whether neutralising ADAM19 activity 0 promoted ADAM19 expression to be elevated in a compen- Chow HFD satory manner in livers from obese and T2D mice. We per- Figure 2: Elevated TNF-𝛼 mRNA levels in the livers of mice on a formed ADAM19 immunohistochemistry on livers of HFD- ∗ high fat diet for 12 weeks. 𝑝 < 0.05; 𝑛=14–17 mice/group. fed mice administered either rabbit IgG control antibody or ADAM19 neutralising antibody. We found that the level of expression did not differ between mice administered rabbit IgG control or ADAM19 neutralising antibody (Figure 8). administered with ADAM19 neutralising antibody had lower Therefore, neutralising ADAM19 activity does not result in blood glucose levels by the conclusion of the experiment a compensatory upregulation of ADAM19 protein expression (Figure 6). This finding indicated that neutralising ADAM19 in the livers from obese and T2D mice. activity improved insulin sensitivity in HFD-fed mice, which correlated with weight loss. This novel data suggests that the 3.6. In Vivo siRNA Mediated Knockdown of ADAM19. To ADAM19 metalloproteinase domain may play a crucial role support our insulin sensitivity findings from the ADAM19 in obesity, insulin resistance, and T2D. neutralisation experiments, we then conducted experiments Mediators of Inflammation 5

∗

6 8 5 7 6 4 5 3 4 3 2 expression mRNA 𝛼 2 1 TNF-

ADAM19 mRNA expression mRNA ADAM19 1 (fold change relative to chow) (fold change relative to chow) to relative change (fold 0 0 Chow HFD Chow HFD (a) (b)

∗ Figure 3: Elevated Adam19 (a) and Tnf-𝛼 (b) mRNA levels in gonadal white adipose tissue of mice on a high fat diet for 16 weeks. 𝑝 < 0.05; 𝑛=7-8 mice/group.

(a) (b)

Figure 4: Neutralisation of ADAM19 reduces high fat diet induced obesity. Representative photographs of HFD-fed mice administered with either rabbit IgG (a) or ADAM19 neutralising antibody (b). Mice administered rabbit IgG possessed increased gonadal adipose tissue compared to mice administered ADAM19 neutralising antibody.

using siSTABLE siRNA targeting mouse ADAM19. We support for a role of ADAM19 in the regulation of human demonstrated that we could effectively knock down ADAM19 obesity, insulin resistance, and T2D. This data prompted us to in vitro (Supplementary Figure 1) and in vivo (Supple- further evaluate the role of ADAM19 using our in vivo obese mentary Figure 2(A)). We highlighted that HFD-fed mice andT2Dmousemodel.WefoundthatADAM19protein administered siSTABLE siRNA targeting ADAM19 possessed expression is elevated in highly steatotic liver and gonadal increased insulin sensitivity compared to HFD-fed mice white adipose tissue of obese and T2D mice. This is extremely administered non-targeting siSTABLE siRNA (Supplemen- interesting as it has been previously reported that murine liver tary Figure 2(B)). has low ADAM19 expression [16]. In our in vivo study, mice fedstandardchowalsohadlowADAM19expression. 4. Discussion We showed that TNF-𝛼 mRNA levels were significantly increased in liver and gonadal white adipose tissue of obese Our group is the first to examine the role of ADAM19 in and T2D mice. TNF-𝛼 is a major proinflammatory cytokine obesity and T2D. We found that high-level expression of implicated in the metabolic syndrome [4, 5], particularly ADAM19 in lymphocytes from the SAFHS cohort strongly insulin resistance [6, 7]. As TNF-𝛼 is a substrate for ADAM19 correlated with the parameters of the metabolic syndrome. In [11–13], we hypothesise that the shedding of TNF-𝛼 by the particular, ADAM19 expression was significantly correlated ADAM19 metalloproteinase domain potentially exacerbates with BMI, relative fat, HOMA-IR, and TNF-𝛼 levels. These the pathogenesis of the metabolic syndrome. In support of significant and novel clinical observations provided strong this hypothesis, we were able to demonstrate in our in vivo 6 Mediators of Inflammation

∗ studies in HFD-fed mice that neutralisation of ADAM19 resulted in a marked reduction in TNF-𝛼 protein in the liver. This TNF-𝛼 protein may be cleaved or cytoplasmic derived. 2 We examined whether neutralising the metalloproteinase domain of ADAM19 would reduce the parameters of the 1 metabolicsyndromeinvivo.Wearethefirsttoshowthat neutralising ADAM19 activity reduces high fat diet induced 0 obesity. In addition, neutralisation of ADAM19 promotes −1 insulin sensitivity in obese and T2D mice. To support this finding, we demonstrated that HFD-fed mice administered −2 siSTABLE siRNA targeting ADAM19 possessed increased insulin sensitivity compared to HFD-fed mice administered % weight loss after administration loss after % weight of ADAM19 neutralising antibody neutralising ADAM19 of −3 nontargeting siSTABLE siRNA. Combined, this novel data Rabbit IgG control ADAM19 neutralising provides evidence that neutralising antibody directed against antibody antibody the metalloproteinase domain of ADAM19 could be a potential therapeutic to alleviate symptoms of the metabolic syn- Figure 5: Elevated weight loss after administration of ADAM19 drome. Currently, there are no specific ADAM19 pharmaco- neutralising antibody. Mice which were administered ADAM19 logical inhibitors available and hence ADAM19 neutralising neutralising antibody lost a significant amount of weight when antibodies appear to be the most realistic available therapy to compared with mice administered the rabbit IgG control antibody. ∗ Mean + SEM; 𝑝 = 0.0126; 𝑛=3mice/group. inhibit the function of ADAM19. Targeting ADAMs with pharmacological inhibitors is being investigated as a potential therapeutic strategy for inflammatory diseases. The highly selective and orally active 16 TACE (ADAM17) inhibitor, Ro 32-7315, has highlighted 14 the reality that metalloproteinase inhibitors may inhibit inflammation induced TNF-𝛼 shedding and can be used 12 safely in humans [27]. Of utmost importance is the fact 10 that administration of TACE inhibitors to humans in clinical 8 trial settings has proven to effectively decrease inflammatory mediators [10]. We used ADAM19 neutralising antibody in 6 our current study which is a clinically relevant therapy. 4 Neutralising antibody that targets TNF-𝛼 is currently used Blood glucose (mmol/L) for rheumatoid arthritis in animal models and humans [28, 2 29]. Currently, there are a limited number of studies which 0 utilise neutralising antibodies that target metalloproteinases 0 min 15 min 30 min 45 min 60 min 90 min 120 min in human disease [30–32]. Therefore, studies like ours are HFD + rabbit IgG very important and may ignite further interest in the area. HFD + ADAM19 neutralising antibody Future studies should aim to examine potential upstream regulators of the increase of ADAM19 protein expression in Figure 6: Neutralisation of ADAM19 improves insulin sensitivity in relation to the metabolic syndrome in humans and mice. HFD-fed mice. Mean ± SEM; 𝑛=3mice/group. A study by Keating et al. (2006) identified that ADAM19 is induced by transforming growth factor beta 1 (TGF-𝛽1) [33]. Human studies have shown that TGF-𝛽1ispositively ∗ correlated with BMI [34] and increased plasma levels of TGF- 50 𝛽1 were reported in T2D [35]. Yadav et al. (2011) observed a significant correlation between TGF-𝛽1 levels and adiposity 40 in humans and rodents, and inhibiting TGF-𝛽1 signalling protected mice from obesity, hepatic steatosis, and diabetes

g of total protein) total g of 30

𝜇 [36]. It would be intriguing to investigate the effect of TGF- 𝛽1 on ADAM19 expression and activation in our mouse 20 model of obesity and T2D. Another interesting potential regulatorofADAM19isfurin.Ourteampreviouslyreported

(pg/mL per 10

𝛼 that the protein furin activates ADAM19 by binding to and cleaving the prodomain of ADAM19 [37]. Relevantly, a TNF- 0 polymorphism in the furin gene (rs17514846) has been shown Rabbit IgG control ADAM19 neutralising antibody antibody to be significantly associated with hypertriglyceridemia and the metabolic syndrome [38, 39]. It would be insightful Figure 7: Neutralisation of ADAM19 reduces liver TNF-𝛼 levels in to ascertain whether furin protein levels are elevated in ∗ HFD-fed mice. Mean + SEM; 𝑝 = 0.05; 𝑛=3mice/group. human patients with the metabolic syndrome and whether Mediators of Inflammation 7

(a) (b)

Figure 8: Neutralising ADAM19 does not affect the expression of ADAM19 in the liver. Representative photomicrographs showing ADAM19 immunohistochemistry performed on livers from HFD-fed mice administered either rabbit IgG control (a) or ADAM19 neutralising antibody (b). ADAM19 staining is cytoplasmic and brown in colour. 200x objective.

the binding of furin to ADAM19 pathogenically activates authors are grateful to the participants of the San Anto- ADAM19 in vivo. nio Family Heart Study. Data collection for the SAFHS Whilst discussing potential regulators of ADAM19, it was supported by a grant from the National Institute for is relevant to also consider what transcriptionally controls Heart, Lungs and Blood (HL045222). A generous donation ADAM19 expression in obesity and T2D. Putative binding from the Azar/Shepperd families paid for the transcriptional sites for the transcription factors Sp1, Sp3, NF-kappaB, and profiles.SOLARissupportedbytheNationalInstitutefor VDRhavealreadybeenidentifiedintheADAM19gene[40]. Mental Health (MH059490). Parts of this investigation were If Sp1, Sp3, NF-kappaB, and VDR are increased in obese and conductedinfacilitiesconstructedwithsupportfromthe T2D subjects, then this could be a mechanism underlying the Research Facilities Improvement Program Grant from the elevated expression of ADAM19 in the metabolic syndrome. National Center for Research Resources, National Institutes of Health, no. C06 RR013556. The AT&T Genomics Com- 5. Conclusion puting Center supercomputing facilities used for this work were supported in part by a gift from the AT&T Foundation Our exciting findings highlight that elevated ADAM19 pro- and with support from the National Center for Research tein expression is associated with the parameters of the Resources Grant no. S10 RR029392. metabolic syndrome in humans and mice. We identified for the first time that neutralising the ADAM19 metallopro- References teinase domain reduced high fat diet induced obesity and improved insulin sensitivity in obese and T2D mice. These [1] S. J. Hunter and W. T. Garvey, “Insulin action and insulin novel results provide evidence that neutralising antibody resistance: diseases involving defects in insulin receptors, signal directed against the metalloproteinase domain of ADAM19 transduction, and the glucose transport effector system,” The could be a potential therapy for anti-obesity agents. American Journal of Medicine, vol. 105, no. 4, pp. 331–345, 1998. [2] A.-P. J. Huovila, A. J. Turner, M. Pelto-Huikko, I. Karkk¨ ainen,¨ Competing Interests and R. M. Ortiz, “Shedding light on ADAM metalloproteinases,” Trends in Biochemical Sciences,vol.30,no.7,pp.413– The authors declare that they have no competing interests. 422, 2005. [3] J. B. M. Jowett, Y. Okada, P. J. Leedman et al., “ADAM28 is Acknowledgments elevated in humans with the metabolic syndrome and is a novel sheddase of human tumour necrosis factor-𝛼,” Immunology and The authors would like to acknowledge the late Dr. Jeremy Cell Biology,vol.90,no.10,pp.966–973,2012. Jowett. Dr. Jowett was a very close collaborator and colleague [4] K.T.Uysal,S.M.Wiesbrock,M.W.Marino,andG.S.Hotamis- for many years and was instrumental in the early stages of this ligil, “Protection from obesity-induced insulin resistance in project. The authors would like to also extend their gratitude mice lacking TNF-𝛼 function,” Nature,vol.389,no.6651,pp. toProfessorCarlBlobel(HospitalforSpecialSurgery,New 610–614, 1997. York) for providing mouse ADAM19 vectors and antibodies [5] G. R. Steinberg, “Inflammation in obesity is the common link directed against mouse ADAM19. This study was supported between defects in fatty acid metabolism and insulin resistance,” by grants from the University of Western Australia (Safety Cell Cycle, vol. 6, no. 8, pp. 888–894, 2007. Net Grant), Diabetes Australia Research Trust (DART), [6]G.S.Hotamisligil,“MechanismsofTNF-𝛼-induced insulin Rebecca L Cooper Medical Research Foundation, and the resistance,” Experimental and Clinical Endocrinology and Dia- Royal Perth Hospital Medical Research Foundation. The betes,vol.107,no.2,pp.119–125,1999. 8 Mediators of Inflammation

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